Project Summary Corneal keratocytes reside between the collagen lamellae in the cornea stroma, and are responsible for secreting ECM components required to maintain normal corneal structure and transparency. Through their interactions with the extracellular matrix (ECM), stromal keratocytes play a central role in fundamental biological processes such as developmental morphogenesis and wound healing. Such interactions also are also important in the field of tissue engineering, where it is necessary to either modulate cell and ECM patterning to produce specific matrix architectures. While much is known regarding the effects of growth factors on keratocyte differentiation on rigid 2-D rigid substrates, corneal keratocytes reside in a complex ECM environment in vivo, which includes a combination of mechanical, topographical and biochemical cues. ECM stiffness, topography and protein composition have all been shown to modulate keratocyte differentiation, contractility and patterning. However, there are still significant gaps in our knowledge of how multiple stimuli are integrated to produce specific cell phenotypes. The overall goal of this proposal is to develop and test novel 2D and 3D experimental platforms that will allow us to systematically incorporate combinations of both soluble and non-soluble cues. In Specific Aim 1 we will determine how substrate elasticity modulates the keratocyte response to soluble biochemical cues by fabricating tunable polyacrylamide substrates over a range of mechanical stiffness values and using traction force microscopy, quantitative immunofluorescence imaging and biochemical assays to evaluate changes in keratocyte differentiation. In Aim 2 we will determine how substrate topography and composition modulates the keratocyte response to biochemical and biomechanical cues using substrates with aligned fibrillar collagen combined with other key ECM proteins. In Aim 3 we will determine how substrate dimensionality (2-D versus 3-D) modulates the keratocyte response to biochemical, biomechanical and topographic cues by fabricating novel 3D sandwich constructs that present these cues to both the ventral and dorsal surfaces of cells. These new experimental platforms can be used to identify, isolate, and investigate the role of key biophysical signaling pathways on the differentiation of keratocytes (e.g. quiescent, migratory, regenerative and fibrotic phenotypes). Knowledge obtained could aid in the development of targeted anti-fibrosis therapies, as well as guide tissue engineering approaches to developing stromal tissue replacements. Importantly, the experimental models developed could have broad application in other fields where biophysical cues and mechanotransduction are known to have a profound impact on cell patterning and behavior, such as developmental and cancer biology.